Precursor cells have become a central interest in medical research. On the one hand, precursors can replace cells that are senescent or damaged by injury or disease, and on the other hand, these cells represent an ideal model for studying development and differentiation and the factors influencing these processes. Employing conventional cell lines for these studies has the disadvantage that individual cell lines may not be fully representative of the complex biology of an intact organism. Moreover, even repeating the tests in multiple cell lines does not reproduce or account for the complex interactions among cells and tissue that occur in an organism.
Efforts have been made for a couple of years to employ permanent cultures of totipotent/pluripotent embryonic stem (ES) cells for the detection of embryotoxic and mutagenic substances and for the preparation of tissue grafts. ES cells can differentiate in vitro in embryo-like aggregates, so-called embryoid bodies (EBs), derivatives of all three germ layers, i.e. mesoderm, ectoderm and endoderm. Thus, embryoid bodies are particularly suited for teratogenic/embryotoxicological studies as well as identification of cell type and tissue promoting factors, and as precursors for implant tissue for the treatment of damaged organs such as infarcted heart. Several protocols for the in vitro production of EBs have been described.
For example, WO02/051987 describes a protocol to obtain embryoid bodies in which the manufacturing takes place preferably with the “hanging drop” method or by methylcellulose culture (Wobus et al., Differentiation 48 (1991), 172-182).
Alternatively to this, spinner flasks (stirring cultures) are described as culture method. Therefore, the undifferentiated ES cells are introduced into stirring cultures and are mixed permanently according to an established procedure. Therefore, 10 million ES cells are introduced into 150 ml medium with 20% FCS and are stirred constantly with the rate of 20 rpm, wherein the direction of the stirring motion is changed regularly. 24 hours after introduction of the ES cells an extra 100 ml medium with serum is added and thereupon 100-150 ml of the medium is exchanged every day (Wartenberg et al., FASEB J. 15 (2001), 995-1005). Under these culture conditions large amounts of ES cell-derived cells, i.e. cardiomyocytes, endothelial cells, neurons etc., depending on the composition of the medium, may be obtained. The cells are selected by means of the resistance gene either still within the stirring culture or after plating, respectively. Recently, international application WO03/004626 described a method for generating large numbers of embryonic stem (ES) cell-derived tissue, wherein the guiding design is preventing EB aggregation by encapsulation of individual or multiple ES cells, for example in the form of agarose microdrops. Using this measure, a high EB efficiency has been reported in so-called encapsulated stirred culture. However, all of those methods are cumbersome and/or do not provide sufficient amounts of embryoid bodies suitable for example for high throughput screening (HTS) assays.
Thus, there remains a need for reliable, easy and cost-effective methods which are capable of providing EBs in sufficient quality and quantity. The solution to this technical problem is achieved by providing the embodiments characterized in the claims, and described further below.